Understanding the biology ofC. elegansrelies on identification and analysis of
essential genes, genes required for growth to a fertile adult. Approaches for
identifying essential genes include several types of classical forward genetic screens,
genome-wide RNA interference screens and systematic targeted gene knockout. Based on
most estimates made from screening results thus far, from 15–30% ofC. elegansgenes
appear to be essential. Genetic redundancy masks some essential functions and pleiotropy
of many essential genes poses a challenge for a full understanding of their functions.
Temperature sensitive mutations are valuable tools for studies of essential genes, but
our ability to analyze essential genes would benefit from development of new tools for
conditional inactivation or activation of specific genes.

1. Overview

Understanding the biology of C. elegans relies on identification and analysis of
essential genes. An essential gene is defined here as a gene necessary for growth to a
fertile adult. Some essential genes were identified fortuitously, but most essential
genes have been identified through mutant screens designed specifically to isolate
lethal and sterile mutations. Starting with Sydney Brenner's pioneering genetic
analysis, many C. elegans geneticists have sought to understand the nature of the genome
by estimating the fraction of the genome devoted to essential functions. Based on the
frequency of X chromosome lethal mutations, Brenner estimated that the C. elegans genome
had about 2000 essential genes (Brenner, 1974). Inclusion of sterile
mutations raised this estimate to about 3000, and a number of screens for lethal and
sterile mutations in specific genomic regions in the 1970s and 80s yielded gene
frequencies raising the upper limit of this estimate to as high as 30% of the genome or
about 5700. (See Herman, 1978 and Johnsen and Baillie, 1997) for
references and discussion. Also see (Feichtinger, 1995, for alternative
statistical analyses arriving at an estimate of about 8500.) With the sequencing of the
genome and the development of methods to target specific genes, it is now within our
power to obtain loss-of-function mutations in every gene in the worm, and efforts are
under way to do just that. However, even with this new ability to systematically knock
out genes, many essential functions can remain cryptic due to genetic redundancy or to pleiotropy.

2. Types of essential genes

There are three types of mutations that identify essential
functions: zygotic lethal mutations (lethals), maternal-effect lethal mutations
(maternal-effect lethals) and sterile mutations (steriles). Zygotic lethals prevent the
development to adult of individuals homozygous for the mutation. Zygotic lethals are broadly
categorized, based on the time of developmental arrest, as embryonic or larval lethals.
Maternal-effect lethals are a special class of sterilizing mutations that prevent the
development of the progeny of hermaphrodites homozygous for the mutation. Such mutations
define genes whose expression in the mother is required for embryonic development. Sterile
mutations prevent the production of fertilized eggs by individuals homozygous for the
mutation. Sterility could arise due to defects in germline development, somatic gonad
development, oogenesis, spermatogenesis, ovulation or fertilization.

3. Methods for identifying essential genes

Classically, most lethal and sterile mutations have been identified by
random mutagenesis followed by either of two types of screens: genome-wide screens for
conditional lethals, such as temperature-sensitive mutations, and screens for
non-conditional lethals and steriles in particular genomic regions for which balancers are
available. More recently this approach has been augmented and, to a certain extent, replaced
by approaches that target individual genes for knockdown or knockout. Two different
large-scale screening methods are being used: systematic RNA interference (RNAi) and
PCR-based screens for intragenic deletions after mutagenesis.

3.1. Classical mutant hunts

Genome wide screens for temperature-sensitive embryonic lethal mutations carried out in the late
1970s and early 80s provided the first stocked lethal mutants for studies of development.
Genes identified in these early screens are named either emb (arrest in embryogenesis) or
zyg (zygote defective) depending on the lab where the work was done, but operationally the
screens were identical. Their analysis provided a foundation for understanding the
complexity of lethal phenotypes as well as insight into the relative contributions of
maternal vs. zygotic gene expression to the developmental process (see Wood, 1988
for references and discussion). Conclusions from the work were that many genes are required
at multiple times in development and maternal gene expression plays a major role in
embryogenesis. Temperature-sensitive lethal mutations are extremely valuable because they
can be used to analyze essential genes with roles in multiple processes and are discussed
more below.

Non-conditional lethal and sterile mutations must be maintained in the
heterozygous condition, requiring constant selection to avoid loss of the mutation. This
problem can be partially overcome by using closely linked visible markers to “balance” the
lethal mutation. Because of this, early screens for lethal and sterile mutations were
limited to small regions of the genome with convenient visible markers (e.g., Rogalski et
al., 1982; Rose and Baillie, 1980). However, the development of
free duplications and crossover-suppressing chromosomal rearrangements (balancer
chromosomes) allowed easy maintenance of lethals and steriles, thus making it feasible to
carry out large-scale screens for non-conditional lethal mutations (see Edgley et al.,
1995 for a detailed discussion of balancer chromosomes). Such screens are
restricted to regions of the genome for which balancers are available. However, because such
balancers cover much of the genome it has been possible to collect mutations representing
more than 860 essential loci by this method (for example Clark et al., 1988;
Gonczy et al., 1999; Herman, 1978; Howell et al., 1987;
Howell and Rose, 1990; Johnsen and Baillie, 1988; Johnsen and
Baillie, 1991; Kemphues et al., 1988; McKim et al., 1988; McKim et al., 1992; Meneely and Herman, 1979; Rogalski
et al., 1982; Rose and Baillie, 1980; Rosenbluth et al., 1983; Sigurdson et al., 1984; Stewart et al., 1998; Zetka and
Rose, 1992). Genes identified in these screens are named let (lethal), ooc
(oocyte defective), mel (maternal-effect lethal), and spe (spermatogenesis defective) or fer
(fertilization defective — identical to spe mutants in phenotype). For mel and spe mutants
it has been possible to develop genome-wide screens for non-conditional mutations in the
absence of balancer chromosomes. In these screens, F1 progeny of mutagenized hermaphrodites
are picked singly onto plates and the phenotypes are scored in the F2. In the case of
spermatogenesis defective mutations, the homozygotes are identified as hermaphrodites that
produce only unfertilized eggs and whose embryo production can be rescued by mating to wild-type males (for example
Argon and Ward, 1980; L'Hernault et al., 1988; Ward and Miwa, 1978). In the case of mel mutations, the homozygous
mutant animals are identified in the genetic background of an egg-laying defective mutation
(Kemphues et al., 1988; Priess et al., 1987). Because egg-laying
defects allow larvae to hatch inside the mother, killing her at a young age, mutations that
block production of larval progeny allow survival of the F2 animals into old age.
Maternal-effect lethal mutations can be distinguished from sterile mutations by the
accumulation of fertilized but unhatched eggs in the uterus. Unfertilized eggs do not form
light-refracting eggshells.

3.2. RNAi

Identification and analysis of essential genes has been
greatly facilitated by RNA interference (RNAi; see Reverse genetics).RNAi leads to
gene-specific mRNA degradation and is triggered by double stranded RNAi specific for the
target gene. The double stranded RNA can be delivered by microinjection, by uptake of dsRNA
by worms in solution (soaking), or by feeding worms bacteria engineered to synthesize the
RNA of interest. Whole genome screens have been carried out by RNAi feeding in a wild-type
genetic background (Fraser et al., 2000; Kamath et al., 2003)
and in the rrf-3 sensitized genetic background (Simmer et al., 2003). Because
RNAi is susceptible to false negatives and often gives incompletely penetrant phenotypes
(see below), it can only provide a minimum estimate for essential genes in the genome.
Although the screens in the rrf-3 genetic background were more sensitive for viable RNAi
phenotypes, they identified slightly fewer essential genes than found in the wild-type
background. Based on RNAi results in the wild-type background, the minimum number of
essential genes is about 1750. (1170 genes with lethal and sterile RNAi phenotypes adjusted
for a false negative rate of 22% and 86% genome coverage; Kamath et al., 2003) Screens have also been carried out using RNAi administered by injection or
soaking worms in dsRNA solutions (Gonczy et al., 2000; Maeda et al., 2001; Piano et al., 2000; Piano et al., 2002).
Comparison of these results to each other and to results from classical genetic screens has
revealed some limitations of RNAi for discovering gene function: some genes appear to be
insensitive to RNAi and outcomes can vary. Some essential genes will give no RNAi
phenotypes, give very weak phenotypes or give phenotypes other than lethality or sterility.
Furthermore, the efficiency of RNAi differs somewhat with different techniques for
introducing the dsRNA triggers (Piano et al., 2002) and even from trial to
trial with the same technique (Simmer et al., 2003). Another limitation, not
restricted to RNAi, is genetic redundancy (discussed below and see Gene duplications and genetic redundancy in C. elegans).

Two approaches have been used to generate targeted loss-of-function mutations by intragenic deletion. Common to both
is the PCR-based screening of populations of randomly mutagenized worms to identify rare
deletions in targeted genes. The worms carrying the deletions are enriched and eventually
isolated by sibling selection schemes. Initially, transposon insertions in targeted genes
were isolated in the expectation that the insertions would be mutagenic (Rushforth et al.,
1993), but because of a high frequency of silent insertions (Rushforth and
Anderson, 1996; Rushforth et al., 1993) a two-step process of
first isolating insertion-bearing worms and then screening populations of these worms for
imprecise excision events was developed (Zwaal et al., 1993). Chemical
mutagenesis, which proved to be more efficient, has now replaced the use of transposon
excision for generating deletions (Jansen et al., 1997). The procedure is
labor-intensive, and not efficient for a single target gene. Therefore, two groups have
organized large scale efforts and are now generating intragenic deletions based on requests
from members of the research community: the C. elegans Gene Knockout Consortium
and the National Bioresource Project for the Experimental Animal C. elegans
Based on results to date, about 1800 genes
have been knocked out with 20-24% being essential (Shohei Mitani, Don Moerman and Bob
Barstead, personal communications). Projecting the higher frequency to the whole genome
(19,727 CDS; Wormbase, release WS136, 12/3/05) yields an estimate of 4645
essential genes. However, this is likely to be inaccurate because gene knockouts are not
being done randomly, but rather are biased toward genes with known functions in other
organisms, or towards genes with known mutant or RNAi phenotypes.

4. Redundancy of essential genes

Many genes are members of multigene families and members of these families can often
have redundant functions. In rare cases where nucleotide sequence is highly conserved, RNAi
with a single trigger molecule can target multiple family members (e.g., Colombo et al.,
2003). If the number of family members is small, it is possible to reveal
redundancies by double or triple knockout or knockdown (e.g., Schubert et al., 2000; Gotta and Ahringer, 2001). Redundant pathways, in which different
processes can accomplish the same essential function, are more cryptic and are not likely to
be detected except by fortuitous double mutant combinations or screens for synthetic lethal mutations (enhancers).
See Gene duplications and genetic redundancy in C. elegans for further consideration of redundancy and
Genetic enhancers for further discussion of enhancers.

5. Pleiotropic essential gene phenotypes

Many essential genes function repeatedly during the life of the worm and can have different
essential roles in different tissues. (See Genetic mosaics) However, null mutant phenotypes
often reveal only one essential role, leading to incomplete understanding of the gene's
role. A clear example is the Notch family member glp-1. Null mutations in glp-1 are sterile
mutations. Analysis of the null alleles revealed that glp-1 was required for germline
proliferation (Austin and Kimble, 1987). Expression of glp-1 during oogenesis
is also required for cell fate specification in the early embryo. However, this function is
masked in animals homozygous for the null allele by the failure of these animals to produce
embryos. The embryonic role of glp-1 was revealed by the recovery of e2072, a special
non-conditional allele that did not affect germline proliferation, and three
temperature-sensitive mutations (Priess et al., 1987). Analysis of these
mutations revealed that signaling through the GLP-1 protein plays major roles in cell fate
specification at multiple times in the early embryo (Hutter and Schnabel, 1994; Mello et al., 1994; Moskowitz et al., 1994; Priess et
al., 1987). Clearly, null alleles generated by targeted deletions will be
inadequate for full understanding of essential gene functions.

The glp-1 example illustrates
not only the problem of pleiotropy but also one solution — the recovery and analysis of
temperature-sensitive mutations. Temperature-sensitive mutations, as discussed above, are
being used effectively for analysis of many essential C. elegans genes. Judicious
temperature shifts can reveal multiple functions for a given gene. For example, shifting
from permissive to restrictive temperature for brief periods early in embryogenesis revealed
distinct roles for glp-1 at the four-cell and the 12-cell stage (Mello et al., 1994). Therefore the continued screening for temperature-sensitive lethal mutations
(e.g., Encalada et al., 2000; Golden et al., 2000; O'Connell et
al., 1998) will provide reagents for specific analyses that often benefit the
community at large. The relative rarity of temperature-sensitive mutations and the extreme
difficulty of targeting such mutations to specific genes restricts their utility.

Another
solution to the pleiotropy problem, conditional gene expression, used effectively in other
genetic models like fruit flies and mice, has not yet been developed for C. elegans. In this
approach, transgenes that have conditional promoters driving the gene of interest or that
have strategically-placed recombination sequences are transformed into null mutant
backgrounds. These constructs can be designed for gene activation or inactivation in
specific tissues, at specific developmental times or in response to drug applications.
Systems for conditional gene expression include the GAL4-UAS system applied effectively in
fruit flies (Duffy, 2002) and the Cre/lox and tetracycline inducible systems
being used in mouse (Gossen and Bujard, 2002). Adaptation of these tools for
C. elegans would greatly facilitate analysis of essential genes.

6. Summary

The number of essential genes in C. elegans as estimated by gene knockout, RNAi and classical genetic
screens appears to be less than 30% of the genome. The combination of RNAi and targeted gene
knockouts ensures the identification of most essential C. elegans genes in the foreseeable
future. Genetic redundancy could mask a significant number of essential functions, but
construction of double and triple mutants or polygenic RNAi has the potential to identify
many of these cases. Studies of null mutations can identify many roles of essential genes,
but some roles of essential genes can be masked by early developmental arrest. Although
temperature-sensitive mutations can overcome this problem, the difficulty of targeting
temperature-sensitive mutations to specific genes makes it desirable to develop additional
tools for conditional inactivation or activation of essential genes.